Ion channels are proteins that control the passive flux of ions through cell membranes by opening and closing (gating) their pores. This proposal seeks to continue work on investigating the fundamental mechanisms by which ion channels gate. The current focus is the large conductance Ca 2+-and voltage activated K+ (BK) channel, which plays a key role in many physiological processes, including control of muscle contraction, regulation of synaptic transmission, and integration of information in neurons. Although much progress has been made towards understanding how Ca 2+ and voltage activate BK channels, and how accessory beta1subunits modulate this activity, many basic questions remain. To work towards answering these questions, cloned BK channels will be expressed in Xenopus oocytes and HEK293 cells, currents will be recorded from single channels with the patch clamp technique, and the data will be analyzed with advanced techniques to determine gating mechanism. The initial hypothesis to be tested is that multiple Ca 2+- and voltage-dependent regulatory mechanisms act jointly to control the opening-closing transitions (gating) of BK channels. To test this hypothesis, the first specific aim will: 1) determine the contributions, including any cooperative interactions, of each of the five proposed Ca 2+-- dependent regulatory mechanisms to the gating of BK channels. Cooperatively it will be resolved by studying the Ca2+-dependent regulatory mechanisms in isolation and in various combinations. The second specific aim will: 2) develop a comprehensive kinetic gating mechanism for BK channels that incorporates all of the known Ca 2+and voltage-dependent regulatory mechanisms and their cooperative interactions. The gating mechanism will be formulated in terms of a large multi-state multi-tiered model that specifies the states, the transitions among the states, the rate constants for the transitions, modulation of the rate constants by Ca 2+ and voltage, and any cooperative interactions involved in the gating. The third specific aim will: 3) determine the mechanism by which beta1subunits modulate the gating of BK channels. This will be done by identifying the particular steps in the kinetic gating mechanism that are modified by beta1subunits. Understanding how ion channels gate their pores, the goal of this research, will facilitate the comprehension, diagnosis, and treatment of diseases associated with defective ion channels (channelopathies).